Need help!!molecular cloning

[bsengez]

Hello everybody,
I have an expression vector about 4600 bp. I cloned my insert in it and made colony PCR. I got very good amplification, so I isolated the plasmids and digested if everything is okey. It should give one band around 360 bp, but now I have 3 bands which are 250,360 and 650. Any idea?

[kiki06]

Hello Bsengez,

you may have:

1) analyzed your restriction sites wrong
2) added an extra restriction enzyme by accident
3) cloned in a restriction site (present in the insert!) - this is a big problem

I would analyze your plasmid and your insert with webcutter.

Cheers

[bsengez]

The restriction sites are okey.
I have cloned it in right positions.
I analyzed my vector in webcutter, thanks for all your attention.
I believed that I haven't added a wrong enzyme, but you know everyone can make mistakes.
Now, I overcome my problems. When I have enough time, I will check it again.

[knockout maker]

Hi there,
I believe it is either a partial digest or star activity. Which enzyme are you using for your test digestions?. Cheers!

[bsengez]

I'm using XhoI, NotI, HindIII, XbaI. There is no star activity with the enzymes and the buffer I used. Thanks, but I have already thought about this possibility.